Kouichi Seki

Relation of Interferon Therapy and Hepatocellular Carcinoma in Patients with Chronic Hepatitis C

Hepatocellular carcinoma, a major cause of death in patients with cirrhosis, is one of the most prevalent malignant tumors worldwide, and its incidence is increasing [1-5]. After isolation of hepatitis C virus (HCV), most patients with chronic hepatitis and those with cirrhosis of unknown origin were found to be positive for anti-HCV [6-8]. Evidence suggests that HCV-related chronic liver disease plays a role in the development of hepatocellular carcinoma [9-13]. A high proportion of patients with hepatocellular carcinoma have anti-HCV, although the prevalence varies geographically. The highest rate of anti-HCV is in southern Europe and Japan, where about 70% of patients with hepatocellular carcinoma are positive for anti-HCV [5]. Interferon has been widely used to treat chronic HCV infection. A series of clinical trials showed that some patients who received interferon had sustained normalization of serum aminotransferase levels and elimination of serum HCV RNA [14-17]. Histologic improvement was also seen in patients who received interferon [14, 18-20]. It is important to determine whether interferon treatment also lowers the incidence of hepatocellular carcinoma in patients with chronic hepatitis C, but the recognized benefits of interferon make a randomized, controlled trial to address this question unethical. We did a retrospective study to compare the incidence of hepatocellular carcinoma in interferon-treated patients with HCV infection and histologically proven chronic hepatitis or cirrhosis with that in historical controls who did not receive interferon. We also examined the relation between response to interferon therapy and incidence of hepatocellular carcinoma. Methods Patients The interferon group comprised 419 consecutive patients with chronic hepatitis C who had undergone liver biopsy 1 to 2 weeks before interferon therapy and had started treatment between January 1992 and December 1993. The control group consisted of 144 consecutive patients with chronic hepatitis or cirrhosis who had undergone liver biopsy between January 1986 and December 1989. All patients had histologically proven chronic hepatitis or cirrhosis (Child-Pugh class A) and were positive for anti-HCV. Interferon Treatment In the interferon group, 176 patients received human lymphoblastoid interferon, 149 received recombinant interferon- 2a, and 94 received recombinant interferon- 2b for 6 months. The median total interferon dose was 480 mU (range, 282 to 800 mU). No patient had received interferon therapy before study entry. Contraindications to interferon treatment included pregnancy, presence of hepatitis B surface antigen, other types of liver disease, autoimmune disease, and any other serious illness. Efficacy of interferon therapy was categorized as follows. Patients with persistent normalization of alanine aminotransferase (ALT) levels during interferon therapy and follow-up were considered to have sustained response. Patients whose serum ALT level was normal at the end of the treatment but increased to an abnormal level after cessation of treatment were considered to have relapse. All other patients were classified as nonresponders. Follow-up Abdominal ultrasonography or computed tomography was performed every 4 to 8 months, and serum -fetoprotein was measured every 2 to 6 months. The diagnosis of hepatocellular carcinoma was confirmed by needle biopsy, by surgically resected tumor specimens, or by typical radiologic findings on hepatic angiography. The starting date of follow-up for patients in the interferon and control groups was defined as the date of liver biopsy. For both groups, the end of follow-up was the development of hepatocellular carcinoma or December 1991 in the control group and the time of the latest abdominal imaging in the interferon group. To detect hepatocellular carcinoma, follow-up examinations were done in 85.4% of controls and 90.7% of patients in the interferon group. The Osaka Cancer Registry was used [21, 22] to determine whether hepatocellular carcinoma had occurred in patients lost to follow-up. This population-based cancer registry has been operating since December 1962 with the cooperation of the Osaka Medical Association, the Department of Health of Osaka Prefecture, and Osaka Medical Center for Cancer and Cardiovascular Diseases. It covers all of Osaka Prefecture, which had a population of 8.6 million in 1995, and registers cases of cancer by using reports from hospitals and clinics and death certificates collected from health centers. One patient in each group who had been lost to follow-up was listed as having hepatocellular carcinoma in the Osaka Cancer Registry. Determination of the Presence of Hepatitis C Virus Antibody and Hepatitis C Virus RNA Hepatitis C virus antibody was measured by first-, second-, or third-generation enzyme-linked immunosorbent assays (Ortho Diagnostics, Tokyo, Japan). Serum HCV RNA was measured by reverse transcription polymerase chain reaction or complementary DNA assay, as reported elsewhere [23, 24]. Assessment of Liver Histologic Findings The histologic findings in liver biopsy specimens were scored by three of the authors in a blinded manner by using two scoring methods. For assessment of histologic staging, fibrosis score (F1 to F3 for chronic hepatitis and F4 for cirrhosis) was used; F1 indicated portal fibrous expansion, F2 indicated portal-portal septa without architectural distortion, F3 indicated portocentral septa with architectural distortion, and F4 indicated cirrhosis [25]. For assessment of histologic grading, a total score of histologic activity (components 1 to 3) of the Knodell histologic activity index was used [26]. Statistical Analysis Patients who did not complete the treatment protocol were included for analysis on an intention-to-treat basis. The chi-square test was used to compare the baseline characteristics of both groups. The Wilcoxon rank-sum test was used to assess a significant difference between tumor sizes in the two groups. The Kaplan-Meier method was used to calculate the cumulative incidence of hepatocellular carcinoma, and the log-rank test was used to compare the cumulative incidence of hepatocellular carcinoma between the groups. To estimate independent risk factors for the development of hepatocellular carcinoma, Cox proportional-hazards regression analysis was used. For analysis, interferon therapy, age, sex, serum ALT level, serum -fetoprotein level, platelet count, histologic staging, and activity scores were used as variables. A P value less than 0.05 was considered statistically significant. Data are expressed as medians and ranges and as risk ratios and 95% CIs. Results Table 1 shows the baseline characteristics of the interferon and control groups. The groups did not differ for age, sex, serum ALT level, or platelet count. In the interferon group, 387 patients (92%) had chronic hepatitis (128 had F1 disease, 138 had F2 disease, and 121 had F3 disease) and 32 (8%) had cirrhosis. In the control group, 124 patients (86%) had chronic hepatitis (30 had F1 disease, 38 had F2 disease, and 56 had F3 disease) and 20 (14%) had cirrhosis (P = 0.005). The proportion of patients with serum -fetoprotein levels greater than 20 ng/mL was higher in the control group (24%) than in the interferon group (15%) (P = 0.011). Table 1. Baseline Characteristics of Interferon-Treated Patients and Historical Controls with Chronic Hepatitis C In the interferon group, 151 patients (36%) had sustained response, 120 (29%) had relapse, and 148 (35%) were nonresponders. In the 143 patients with sustained response, serum HCV RNA was measured during follow-up. Sustained absence of serum HCV RNA was noted in 120 (84%) of these patients. Twenty-one patients could not complete the 6-month treatment protocol because of depression (5 patients), severe general fatigue (4 patients), skin eruptions (2 patients), severe reduction of serum platelet count (1 patient), pulmonary tuberculosis (1 patient), interstitial pneumonia (1 patient), severe nausea (1 patient), ischemic colitis (1 patient), cardiomyopathy (1 patient), hyperthyroidism (1 patient), and hypermenorrhea (1 patient). One patient stopped treatment because of his business, and one patient discontinued treatment after 3 months because hepatocellular carcinoma was diagnosed. Only 1 of the 21 patients who did not complete treatment showed sustained response; all others were nonresponders. Median follow-up was 47.6 months (range, 3.3 to 65.2 months) in the interferon group and 46.8 months (range, 6.9 to 71.6 months) in the control group. During follow-up, hepatocellular carcinoma was found in 19 controls (4 with F2 disease, 8 with F3 disease, and 7 with F4 disease). In the interferon group, 28 patients developed hepatocellular carcinoma during follow-up (2 patients with F1 disease, 5 with F2 disease, 13 with F3 disease, and 8 with F4 disease). A final diagnosis of hepatocellular carcinoma was made histologically in 17 patients in the interferon group (61%) and 11 controls (58%). In 11 patients (39%) in the interferon group and 8 controls (42%), a final diagnosis was made on the basis of typical angiographic findings. The maximum tumor sizes of hepatocellular carcinoma in the interferon and control groups at the time of discovery on ultrasonography or computed tomography were 20 mm (range, 10 to 52 mm) and 24 mm (range, 10 to 50 mm), respectively (P > 0.2). Figure 1 shows the cumulative incidence of hepatocellular carcinoma in the interferon and control groups, estimated by using the Kaplan-Meier method. The 4-year rate of hepatocellular carcinoma incidence was 6.6% in the interferon group and 12.2% in the control group (log-rank test, P = 0.040). Figure 1. Cumulative incidence of hepatocellular carcinoma (HCC) in interferon-treated patients (dotted line) and historical controls (solid line) with chronic hepatitis C. P Cox proportional-hazards regression analysis was performed to identify factors co

“Nonalcoholic steatohepatitis” induced by massive doses of synthetic estrogen

SummaryWe have noticed that functional disorders of the liver characterized by hepatomegaly and an increase in serum gamma-glutamyl transpeptidase develop in patients with prostatic cancer who are placed under longterm therapy with massive doses of estrogen after castration. We performed laparoscopy in six cases of prostatic cancer with hepatomegaly so that we could study the morphology of the liver. Our findings were as follows. In five, the histological features of the liver biopsies were very similar to those seen in alcoholic hepatitis. In spite of this fact, two of the five had no history of alcohol consumption. Furthermore, in one other case, liver damage resembling alcoholic hepatitis developed during abstinence. The findings in these three cases suggested that long-term, massive doses of synthetic estrogen may lead to liver injury similar to alcoholic hepatitis in nonalcoholics. The ultrastructural findings of the liver cells were also suggestive of the adverse effect of treatment. All cases were negative for hepatitis B surface antigen. Recent reports have demonstrated some nonalcoholics with histological features of the liver indicative of alcoholic hepatitis. This particular condition was termed “nonalcoholic steatohepatitis” by Ludwig et al. It is quite likely that synthetic estrogen is also responsible for “nonalcoholic steatohepatitis” when it is used in massive doses.

Efficacy of combination therapy of interferon-α with ursodeoxycholic acid in chronic hepatitis C: A randomized controlled clinical trial

The efficacy of interferon-α therapy in the treatment of chronic hepatitis C is still limited. A combination therapy of interferon-α with ursodeoxycholic acid (UDCA) was tested for its efficacy in the treatment of chronic hepatitis C by a randomized controlled study. Eighty consecutive Japanese patients with chronic hepatitis C were randomly divided into two groups: one group was treated with interferon-α (group A,n=40) and the other with a combination of interferon-α and UDCA (group B,n=40). In both groups, human interferon-α (6 million units per day) was intramuscularly injected daily for 2 weeks and then three times a week for 22 weeks: this 24-week period was followed by 24 weeks of observation. In group B, UDCA was also administered, daily at a dose of 600mg orally, from the beginning of the interferon therapy and administration was continued for 48 weeks. The rates for ALT normalization and clearance of hepatitis C virus (HCV) viremia at the end of the 24-week interferon therapy were similar for groups A and B (58% vs 60% and 55% vs 48%, respectively). At the end of the 24-week follow-up, the sustained normalization rates for ALT levels for the two groups were not different (35% vs 43%), while the rate of clearance was higher in group B (40%) than in group A (23%), but the difference was not significant (P=0.14). The sustained complete response, i.e., HCV RNA negativity at the end of the follow-up, as well as the maintenance of ALT normalization during the follow-up period, was more frequent in group B (38%) than in group A (18%) although the difference was not significantP=0.08). The rate of HCV reactivation after interferon was discontinued was significantly lower in group B (16%) than in group A (59%) (P<0.01). Although this combination therapy did not lead to a sufficiently sustained complete response, it could serve as adjuvant antiviral therapy when a suitable dosage and administration period are determined.

Effect of high doses of synthetic estrogen on lipid metabolism in castrated male rats

The mechanism by which high doses of estrogen influences lipid metabolism was studied with a microtubular blocking agent. Castrated male rats received oral injection daily for 14 days of 3 mg hexestrol in olive oil, or oil alone as controls. About half of the animals in each group were injected intraperitoneally with 4 mg/100 g body weight colchicine 3 hr before they were killed. Hexestrol treatment caused an accumulation of esterified cholesterol in the liver while it decreased those in serum. Triglyceride concentrations slightly decreased in the liver but were unaffected in serum. On polyacrylamide-gel disc electrophoresis, the peaks of high density lipoproteins (HDL) and low density lipoproteins (LDL) were decreased remarkably. Electron microsopic examination of hepatocytes revealed electron-lucent lipid droplets in the cytoplasm.After a colchicine treatment of the control animals, concentrations of esterified cholesterol and triglycerides markedly increased in the liver, while those in serum decreased. Electron microscopic examination of hepatocytes revealed numerous secretory vesicles filled with nascent VLDL. In hexestrol-treated animals, the colchicine treatment was associated with marked decreases in serumesterified cholesterol and triglyceride as seen in the controls. However, there were no further increases of esterified cholesterol in the liver, and the increase of triglycerides was slight. Electron microscopic examination showed less secretory droplets than in the controls.These data suggest that very low density lipoproteins (VLDL) synthesis in the liver of hexestrol treated rats was inhibited. An accumulation of esterified cholesterol with a marked decrease in serum could not be accounted for by the inhibition of lipoproteins secretion, but rather by their enhanced entry into the liver.

Morphological changes of the liver in uremic patients treated with chronic hemodialysis Laparoscopic observations and light and electron-microscopic studies

SummaryIn order to clarify morphological changes of the liver in the uremic state, 16 uremic patients treated with chronic hemodialysis were studied. Biopsy was performed in 14 cases under laparoscopic observation and in two on the occasion of renal transplantation. One uremic patients not being treated with dialysis was also studied for comparison. All biopsy specimens were examined by light and electron microscopy.The liver usually appeared mildly or moderately swollen under laparoscopic. observations, which was considered at least partially due to the enlargement of the hepatocytes. All patients had hepatocytes with an Orcein-negative “ground glass” appearance, in which marked proliferation of smooth endoplasmic reticulum (SER) was found by electron microscopy. Since the patient not being on dialysis also had such hepatocytes, this finding may be characteristic of uremia. With electron microscopy, in addition to proliferation of SER, alteration of mitochondria and rough endoplasmic reticulum (RER) and an increase in cytoplasmic lipid droplets were observed. Hypertrophy of the Golgi apparatus containing electron-dense particles (VLDL) was often found in patients associated with hypertriglyceridemia. Amorphous electron-dense inclusions in microbodies were occasionally observed.Siderosis was observed in nine patients including three having parenchymal siderosis. With electron microscopy, various siderosomes were seen in the cytoplasm of hepatocytes in patients with parencymal siderosis.Conclusively, these histological and ultrastructural features of hepatocytes are rather associated with several metabolic abnormalities in uremia.

Changes in serum hepatic fibrosis markers in biochemical responders to interferon therapy for chronic hepatitis C.

Serum hepatic fibrosis markers (7s domain of type IV collagen, N-terminal peptide of type III procollagen, and hyaluronate) were determined during and after a 6-month interferon treatment of patients with chronic hepatitis C. Changes in these markers were compared among the patients who showed a sustained normalization of serum alanine transaminase (ALT) levels with and without eradication of serum hepatitis C virus RNA (complete responders and biochemical responders) and nonresponders. In the case of complete responders, the serum 7s domain of type IV collagen and the N-terminal peptide of type III procollagen levels decreased at the end and 24 weeks after the end of the treatment. Hyaluronate levels were significantly decreased 24 weeks after the end of the treatment, as compared with those prior to the treatment. During and after interferon treatment, changes in these markers in the case of biochemical responders were nearly the same as those in the complete responders. These results suggest that serum hepatic fibrosis markers decrease in patients with chronic hepatitis C who show a sustained normalization of ALT after interferon treatment, even if serum hepatitis C virus RNA fails to be eradicated.

A case of chronic veno-occlusive disease of the liver

SummaryA case of chronic veno-occlusive disease of the liver manifested by ascites in a 59-year-old Japanese male is described. The patient had not been exposed to pyrrolizidine alkaloids, but had been receiving allopurinol for hyperuricemia, the only drug taken throughout the period in which the hepatic lesion developed. The hepatic veins and inferior vena cava were patent according to angiography. The first biopsy showed histological characteristics of the disease and the following two biopsies demonstrated the progression of the hepatic lesion from central fibrosis to non-portal cirrhosis. Morphometric analysis of the sublobular and central veins in the three serial biopsy specimens demonstrated stepwise decrease of the ratio of the veins per portal triad and stepwise increase of severely obliterative veins.

Hypertrophic and hypoactive smooth endoplasmic reticulum in hepatocytes of uremic patients. A morphometric and biochemical study

SummaryMorphometric analysis of the hepatic endoplasmic reticulum and assays of drug-metabolizing enzymes in microsomes prepared from needle biopsy specimens were performed with samples from uremic patients on chronic hemodialysis and normal controls. The smooth endoplasmic reticulum (SER) morphometrically increased in the uremic patients, whereas the cytochrome P-450 content and the activity of ρ-nitroanisoleO-demethylase per mg microsomal protein decreased in the uremic patients. This condition of SER corresponds to that of the so-called hypoactive hypertrophic smooth endoplasmic reticulum, which could suggest lower activity of hepatic microsomal drug oxidation in uremic patients. On the other hand, the P-450 content and the activity of ρ-nitroanisoleO-demethylase per g liver were not significantly different between the uremic and the normal subjects. This seems to indicate that the capacity of drug oxidation is retained in whole livers of uremic patients. However, since some patients in this study showed markedly low activity of ρ-nitroanisoleO-demethylase per mg microsomal protein and per g liver, lipophilic drugs metabolized in the liver as well as hydrophilic ones eliminated by the kidney should be carefully administered to uremic patients.

Studies on the primary hepatic carcinoma in our clinic

patients had fever and ascites was observed on 16 patients in which bloody ascites was prooved in 10 cases. Jaundice also appeared on 16 patients. Abdominal pain was severe in patients who had remarkable hemorrhage and necrosis of the liver and there were no distinct relations between abdominal pain and weight of the liver, extention and metastasis of the tumor. Ascites was remarkable in cases which had pressure or obstruction around the porta hepatis and cirrhosis of the liver, and tendencies of hypoalbuminemia have been noticed in these cases. Bloody ascites was mainly observed in case which had livers weighed over 2500 grams or in cases which had very extensive tumors. Also all cases that had ascites and died whithin a month showed bloody ascites. Jaundice was not so remarkable in cases which had pressed porta hepatitis but was remarkable in cases which had extensive tumor invasion in the liver. In the cases that showed jaundice, depositions of bile pigments were eminently seen in liver cells and the aspects which were suggestive of the secretion of bile pigments by tumor cells were observed partly. High fever appeared in cases which had marked infiltration of leukocytes combined with infection, hemorrhage and necrosis of the liver. Antibiotics were effective in these cases. However, they were not effective in the most of cases which had slight fever under 38~ and had little complication of infection.